High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation.

High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation.

First published online: 2012-03-10 / 名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年4月27日 森谷隆氏の博士論文として提出された

Indiana University-Purdue University Indianapolis (IUPUI) / A burgeoning area of focus within liver disease research is centered on the concomitant muscle atrophy present in end stage liver disease patients which shows a correlation to severity of hepatic fibrosis and transplant survival outcomes. Of particular interest, nonalcoholic steatohepatitis (NASH) is a form of liver disease that is characterized as the hepatic manifestation of metabolic syndrome. If left untreated, the disease can progress to the state of cirrhosis and hepatocellular carcinoma requiring transplant. Concordant with increasing global prevalence of obesity, NASH is projected to become the leading cause for liver transplants by 2020. Due to a lack of therapeutic options, these patients represent a large unmet medical need in the western world. A major hurdle to therapeutic research is the lack of a quick, reproducible, and cost effective in vivo model that recapitulates the plethora of pathologies and their molecular underpinnings manifested by this disorder. Our studies attempted to validate and expand upon a two-hit model of NASH, which incorporated both the integral comorbidities associated with metabolic challenges of obesity along with liver injury. The two-hit model manifests not only the hepatic morphohistological characteristics of the disease, but also incorporates the obligatory muscle atrophy. To further elaborate on the potential direct link between liver and skeletal muscle and remove any confounding issues associated with the model, in vitro administration of hepatotoxins representing various pathologies associated with liver disease, were used to recapitulate the liver-muscle endocrine signaling that exists in vivo. Our data shows that a variety of hepatoxins can elicit hepatocellular damage which releases factors that inhibits myotube size in vitro. The two hit model also preserves many of conserved molecular underpinnings observed in clinical hepatic fibrosis. Of particular interest, the TGFβ superfamily has been demonstrated to play an important regulatory role in the progression of fibrosis in NASH patients. TGFβ, Activin A, and Follistatin are members of the highly conserved family that are increased in NASH patients. Furthermore, these proteins have a well-studied role in muscle health, regeneration, and mass that has been hypothesized to be conserved between liver and muscle tissues. Surprisingly, novel expression of the myokine and negative regulator of muscle mass Gdf8 (myostatin) was increased in our in vivo model as well. Our studies focused on the molecular interactions of these TGFβ superfamily members and their role on liver disease progression. Through specific inhibition of these proteins (Activin A and Gdf8), we demonstrated that they appear to play key individual roles in the progression of the concomitant muscle atrophy observed in NASH patients. Interestingly, superior efficacy was gained with the treatment of a pan inhibitor of these proteins (Activin A, B, Gdf8 etc.) via a soluble decoy receptor (ActRIIB-Fc), suggesting an additional unaccounted for ligand. Activin B, was found to be increased in two separate in vivo models of liver fibrosis (two-hit model and BDL), has been implicated in regulating muscle mass. Our data suggest a pivotal role for several members of the TGFβ superfamily in NASH associated muscle atrophy. Therapies designed to treat liver fibrosis and the resultant decrements in muscle mass and force must account for these agents which will require pan inhibition of TGFβ superfamily ligands that signal through the ActRIIB receptor.

Die Nicht-alkoholische Steatohepatitis (NASH), gehörig zu der Gruppe der chronischen Lebererkrankungen als eine schwere Form der Nicht-alkoholischen Fettleber-erkrankungen (NAFLD), nimmt in ihrer Prävalenz ständig zu. Gründe dafür sind u.a. eine gesteigerte Nahrungsaufnahme sowie Veränderungen der Nahrungszusammen-setzung. Es kommt zur Ausbildung einer Steatose, die sich unter Mitwirkung verschie-dener Einflussfaktoren zur Steatohepatitis weiterentwickeln kann, wobei die Pathoge-nese noch nicht genau verstanden ist. Die Nicht-alkoholische Steatohepatitis geht oft einher mit Insulinresistenz und starkem Übergewicht. Die Folgen für die Leber sind Funktionseinschränkungen und –verlust, hervorgerufen durch eine massive Akkumula-tion von Triglyzeriden in den Hepatozyten, Entzündungsprozesse sowie einem fibro-tischen Umbau der Leber. Im fortgeschritten Stadium wird eine Lebertransplantation unausweichlich, die jedoch aufgrund des zunehmenden Mangels an Spenderorganen oft nicht möglich ist. Eine Alternative bietet die Transplantation mesenchymaler Stammzellen (MSC). MSC können in vitro in leberzellähnliche Zellen differenziert wer-den und weisen dabei essentielle hepatozytäre Eigenschaften auf, wodurch sie als möglicher Ersatz bzw. als Überbrückungstherapie bis zur Lebertransplantation in Frage kommen. Die vorliegende Arbeit beschäftigte sich mit dieser Fragestellung. Dazu wur-de ein Tiermodell der NASH mittels Methionin-Cholin-defizienter Diät (MCD-Diät) etab-liert und die Transplantation von hepatozytär differenzierten MSC durchgeführt. An-hand spezifischer zellulärer und biochemischer Marker der NASH konnte die Wirkung des Zelltransplantats auf die Empfängerleber analysiert werden. Es hat sich gezeigt, dass die MSC einen anti-inflammatorischen, anti-fibrotischen und pro-proliferativen Einfluss auf das Empfängerparenchym hatten und somit zur Verbesserung der Symptomatik der NASH beitrugen.

Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant inter individual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mouse model, indicating attenuation of the activity of Cyp1a2 and Cyp2c29, respectively. Decreased mRNA expression of Cyp1a2 and Cyp2c29, as well as an overall decrease in CYP protein expression, was found in the diabetic NASH mice. Overall, these data suggest that the diabetic NASH model only partially recapitulates the human ex vivo CYP alteration pattern. Therefore, in vivo determination of the effects of NASH on CYP activity should be conducted in human, and more appropriate models are required for future drug metabolism studies in NASH.

The human genome contains slightly more than 20 000 genes that are expressed in a tissue specific manner. Transcription factors play a key role in gene regulation. By mapping the transcription factor binding sites genome-wide we can understand their role in different biological processes. In this thesis we have mapped transcription factors and histone marks along with nucleosome positions and RNA levels. In papers I and II, we used ChIP-seq to map five liver specific transcription factors that are crucial for liver development and function. We showed that the mapped transcription factors are involved in metabolism and other cellular processes. We showed that ChIP-seq can also be used to detect protein-protein interactions and functional SNPs. Finally, we showed that the epigenetic histone mark studied in paper I is associated with transcriptional activity at promoters. In paper III, we mapped nucleosome positions before and after treatment with transforming growth factor  β (TGFβ) and found that many nucleosomes changed positions when expression changed. After treatment with TGFβ, the transcription factor HNF4α was replaced by a nucleosome in some regions. In paper IV, we mapped USF1 transcription factor and three active chromatin marks in normal liver tissue and in liver tissue of patients diagnosed with alcoholic steatohepatitis. Using gene ontology, we as expected identified many metabolism related genes as active in normal samples whereas genes in cancer pathways were active in steatohepatitis tissue. Cancer is a common complication to the disease and early signs of this were found. We also found many novel and GWAS catalogue SNPs that are candidates to be functional. In conclusion, our results have provided information on location and structure of regulatory elements which will lead to better knowledge on liver function and disease.

Correct dosing in pharmacotherapeutics is based on the idea that too much of a drug will cause toxicity, while too little will result in failure to elicit the desired response. A major factor in the ability of a patient to handle any dose of a drug is the capacity to metabolize and eliminate that drug from the body. For the vast majority of drugs, the liver plays a key role in determining the rate at which drugs are eliminated. First, drugs must be taken up across the cell membrane into hepatocytes by uptake transporters. Once inside the hepatocyte, biotransformation enzymes metabolize and conjugate the drug to a more water-soluble compound, the distribution of which is more easily controlled. These water-soluble metabolites are then transported out of the hepatocyte by additional drug transporters either into bile for elimination, or back into the blood.More than 2 million severe adverse drug reactions occur in the US each year and often result from interindividual variation in the ability to metabolize and eliminate drugs. This number does not include medical errors, but rather circumstances where an individual is unable to handle the standard dose of the correctly prescribed drug. Although genetics plays an important role, the greatest source of variation comes from other environmental factors such as disease states. Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that comprises a spectrum of histopathologies that range from simple steatosis to the more severe steatohepatitis. Specifically, nonalcoholic steatohepatitis (NASH) has become one of the leading causes for liver transplantation in the United States, and thus clearly become a considerable burden to the U.S. healthcare system.It is not known whether the capacity of the liver to metabolize and excrete drugs is altered in patients with NASH. Because the liver plays such a critical role in drug metabolism and disposition, any disease state that disrupts or modifies these functions will alter the fate of a given drug within the body. It is therefore very likely that the ability of the liver to metabolize and excrete clinically relevant drugs is compromised in NASH patients.

No description available.

nÃo hà / IntroduÃÃo: A esteatohepatite nÃo alcoÃlica (NASH) à um evento adverso do quimioterÃpico irinotecano extremamente relevante, pois associa-se a uma maior mortalidade apÃs ressecÃÃes hepÃticas. Atà o momento nÃo hà na literatura um modelo experimental bem estabelecido de NASH induzido por irinotecano. Objetivo: Estabelecer um modelo experimental de NASH induzida pelo irinotecano em camundongos que simule os aspectos histopatolÃgicos encontrados na clÃnica. Estudar os mecanismos e mediadores envolvidos na sua fisiopatologia incluindo citocinas inflamatÃrias, estresse oxidativo e participaÃÃo da translocaÃÃo bacteriana intestinal. MÃtodos: Camundongos machos do tipo Swiss, pesando entre 25 a 30g,foram divididos em grupos experimentais (n=8-10) e foram injetados com soluÃÃo salina (5 mL/kg, i.p.) ou Irinotecano nas doses 25, 50, 75 or 100 mg/kg, i.p, trÃs vezes por semana em dias alternados. Os animais foram entÃo pesados e sacrificados ao fim das semanas 1, 3, 5, 7 e 9. No sangue foram dosados os nÃveis de ALT e AST, leucograma, proteÃnas totais e realizada hemocultura da veia porta e do plexo ocular. Nas amostras do tecido hepÃtico, avaliaram-se os nÃveis de lipÃdeos totais, mieloperoxidase, malonaldeÃdo e glutationa (GSNP) seguida da anÃlise histopatolÃgica realizada apÃs preparaÃÃo da lÃmina pelos mÃtodos HE e Masson para avaliar e graduar os critÃrios histolÃgicos da NASH. As amostras de duodeno foram examinadas histologicamente (HE) para graduar a mucosite. ImunohistoquÃmica para IL-1, IL-18, NOSi, TNFα e TLR-4 foi realizada tanto nas amostras hepÃticas como duodenais. A anÃlise estatÃstica foi realizada empregando o teste t de Student para variÃveis nÃo pareadas ou o teste Mann-Whitney quando indicado. Resultados: A dose de 50mg/kg de irinotecano trÃs vezes por semana i.p. (IRI 50) se associou com perda de peso, leucopenia, hepatomegalia, diminuiÃÃo das proteÃnas plasmÃticas e aumento das enzimas hepÃticas (ALT e AST). IRI 50 alterou os parÃmetros de estresse oxidativo, elevando a expressÃo de malonaldeÃdo e diminuindo GSNP com inÃcio na primeira semana do experimento. IRI 50 tambÃm se associou a todas as alteraÃÃes histolÃgicas da NASH (esteatose, infiltraÃÃo neutrofÃlica e tumefaÃÃo celular e fibrose) analisadas na sÃtima semana quando comparado com controle salina. Nas amostras duodenais, observamos alteraÃÃes histolÃgicas de mucosite severa a partir da terceira semana. Houve tambÃm em IRI 50 aumento da imunoexpressÃo de IL-1, NOSi e TLR-4 tanto nas amostras do fÃgado como nas do duodeno na semana 7. Nas hemoculturas de sangue portal e orbitÃrio identificou-se o crescimento de enterobactÃrias gram-negativas desda a primeira semana do experimento. ConclusÃo: A administraÃÃo de irinotecano 50mg/kg i.p. trÃs vezes por semana por sete semanas consecutivas à um modelo experimental que reproduz todas as alteraÃÃes histopatolÃgicas da NASH induzida por irinotecano observadas na clÃnica. Existem evidÃncias da participaÃÃo concomitante das citocinas inflamatÃrias e do estresse oxidativo na patogÃnese da NASH em nosso modelo. A translocaÃÃo bacteriana intestinal com bacteremia portal por gram-negativos pode ser o insulto primeiro e o elo de conexÃo comum entre os diversos fatores fisiopatolÃgicos. / Introduction: The nonalcoholic steatohepatitis (NASH) is a relevant adverse effect of the chemotherapy with irinotecan, since it has been associated with increased surgical mortality after hepatic resections. So far there are no reports in the literature of a well established experimental irinotecan-induced NASH model. Aim: Develop an experimental model of irinotecan-induced NASH in mice that simulate the full histological alterations found in the clinical practice. Study the possible mechanisms and mediators involved into its pathophysiology including inflammatory cytokines, oxidative stress and the participation of intestinal bacterial translocation. Methods: Male swiss mice weighting between 35 and 30g were divided into experimental groups (n=8-10) and were injected with saline (5 mL/kg, i.p.) or irinotecan in the following dosages: 25, 50, 75 or 100 mg/kg, i.p, three times a week every other day. Mice were weighted and killed at the end of the weeks 1, 3, 5, 7 and 9. Measurements of ALT and AST, leukocytes, total proteins were performed in blood samples as well as cultures of portal vein and ocular plexus blood. In the hepatic tissue samples, total lipids, myeloperoxidase, malonaldehyde and nonproteic sulfidryl groups (NPSH) levels were analyzed, followed by histological evaluation, after slide preparation through HE and Masson methods. Duodenal samples were examined histologically for mucositis grading. Immunohistochemistry analysis for IL-1, IL-18, NOSi, TNFα e TLR-4 were performed in both hepatic and duodenal tissue samples. Statistical analysis was carried out through Studentâs t test or Mann-Whitney test, as indicated. Results: The dose of 50mg/kg of irinotecan three times a week i.p. (IRI 50) associated to weight loss, low white cell count, hepatomegaly, decreased total plasma proteins and elevated hepatic enzymes (AST and ALT). IRI 50 altered oxidative stress parameters, elevating elevating malonaldehyde and decreasing NSPG levels at hepatic tissue starting at week 1. IRI 50 also was associated to the full histological changes found in NASH (steatosis, neutrophilic infiltration and hepatocyte balloning) when analyzed at the seventh week and compared to saline group. The duodenal samples showed severe mucositis changes starting at week three. It was also demonstrated elevated immunostaining for IL-1, iNOS and TLR-4 in both liver and duodenal samples at week seven. The cultures were positive for gram negative intestinal bacteria in portal and orbital blood since the first experimental week. Conclusion: The administration of the irinotecan 50mg/kg i.p. three times a week for seven consecutive weeks is an experimental model that reproduces all the histological changes found in the irinotecan-associated NASH found in clinical practice. Concomitant participation of inflammatory cytokines and oxidative stress were found to play a role in NASH pathogenesis in this model. Intestinal gram-negative bacterial translocation leading to portal bacteremia may represent the very first hepatic insult and the connection between the pathogenic factors.